The receptors for corticotrophin-releasing factor (CRF) play central roles in the physiology of the pituitary gland and central nervous system. The molecular cloning of two CRF receptor subtypes that are the product of two different genes revealed that these receptors play an important role not only in the neuro-endocrine system but also in the cardiovascular system. The applicant proposes to study the structural features of the CRF receptor subtype, CRF-Ra, that is responsible for the neuroendocrine regulation of corticotroph function. The applicant will characterize the structural features that determine CRF receptor function by assessing the role of receptor glycosylation, disulfide bridge formation, and by constructing hybrids with the PTH/PTHrP receptor. Preliminary studies have shown that an insertion of 29 amino acids in the first cytoplasmic loop uncoupled the receptor from G proteins. Therefore, the applicant will focus efforts to characterize the role of this loop in G protein coupling. Molecular cloning of the chicken receptor during the first funding period of the project revealed that evolutionary changes in its primary sequence impact on the receptor's ability to bind CRF and CRF-like ligands. The applicant therefore will continue efforts to analyze evolution of the receptor by cloning fish and Xenopus CRF receptors, and expressing them to characterize their binding and signaling properties.